Microorganisms from insects, bacterial and viral pathogens are harmful agents which threaten human and animal health and greatly increase the costs and the financial risks of food production. These threats are particularly true in farms and aquaculture and where “livestock” are grown in high intensity production facilities. The detection and identification of these agents by the genomic material via PCR, RT-PCR (wherein PCR means polymerase chain reaction and RT-PCR means reverse transcription PCR) and gene sequencing are now well established and are proving useful in strategies for the detection and control of these agents. However, the application of these methods for direct detection of nucleic acids contained in aquacultural, agricultural, environmental and clinical samples has often proven inefficient and most often not possible.
Many reasons for this detection inefficiency are known. These include the fact that the target nucleic acids are frequently entrapped or compartmentalized inside the microorganism and inside the tissues of the host. This compartmentalization can render the genomic material of the microorganism (“target nucleic acids”) unavailable for direct detecting. As consequence, a method to release and solubilize target nucleic acids is needed for detection. In known methods, target nucleic acids are comingled both with inhibitors and with large amounts of host DNA and RNA contained in the cells and tissues, complicating detection of target nucleic acids.
Animal, marine and environmental samples are further complicated by solid sediments, colloids, emulsions, soluble and insoluble salts, biopolymers, biodegraded debris, inert materials and contaminating industrial and natural chemicals in the samples. The heterogeneity and complexity of these samples can inhibit or invalidate direct nucleic acid detection methods. Sample coloration, also can obstruct test results when colors are part of the analysis or detection method.
Direct detection of microorganisms and pathogens is further complicated in many instances, as samples contain only a few copies of the target genomic material. Furthermore, in these samples, the target genomic material is frequently present with large numbers of genomic materials from both the host and non-target organisms present in the samples. Because of the low target copy number and the high concentration of background genomic material and inhibitors, the sensitivity and specificity of direct detection approaches such as PCR, RT-PCR and immunoassays are subject to chemical interferences and inefficiencies in detection.
To circumvent these problems, some form of sample treatment is required to concentrate, purify and free the target genomic material from interfering materials. Many sample preparation approaches have been developed to obviate the inaccuracies and false results due to the loss of sensitivity and compromised specificity resulting from genomic compartmentalization, inhibitors and inefficient genomic detection.
Generally, these treatment processes involve: i) tissue disruption and cell lysis to release the target genomic material, ii) adsorption of the genomic material onto a solid support such as membranes or adsorbent materials, iii) washing the adsorbed genomic material free of contaminants, and iv) releasing the genomic material from the solid support for detection.
Although the methods described above have proven useful for the detection and identification for detection of genomic materials, they still suffer from the need for multistep processes to purify and isolate the genomic material prior to analysis. Such processes not only entail use of supporting equipment and materials, (e.g. centrifugation, membrane filtration and/or chemical precipitation) they may also require toxic chemicals and solvents to stabilize the genomic material and to aid in the release of the genomic material from the solid supports. These methods not only add complexity, cost and time to the analysis but may also need to be practiced in the laboratory using vacuum or electrical equipment.
As a consequence, there is still a need for a method for detection of harmful agents such as microorganisms, including bacterial and viral pathogens, that can be practiced in the field, without need for laboratory vacuum or electricity, is rapid and simple to use, does not require non-toxic chemicals, and is low cost and can be used for the rapid detection and identification of pathogens based on diagnostic gene sequences. By “field” it is meant, for example, a site for growth or production or processing of food products, including but not limited to farms (both aquaculture and agriculture), food processing and packaging sites, and the like.
To monitor and control detrimental microorganism and to control diseases they cause, there is further a need for a simple sample processing technology which can be used in the field to collect, process and recover genomic materials from samples in which microorganisms be found for analysis of a target nucleic acid of the microorganism.
The present invention meets these needs.